"Angiogenesis" refers to the growth of new blood vessels, or "neovascularization", and involves the growth of capillaries composed of endothelial cells. Angiogenesis is an integral part of many important biological processes, including cancer cell proliferation, inflammation, wound healing, the menstrual cycle, and fetal development. New blood vessel formation is required for the development of any new tissue, whether normal or pathological, and thus represents a potential control point in regulating many disease states, as well as a therapeutic opportunity for encouragement of the growth of normal tissue.
One paradigm for this process is encountered early in embryonic development when primordial cells form "blood islands," the cells of which give rise to capillaries, which in turn form the larger veins and arteries in the process of vasculogenesis. However, once the vascular system is formed, usually by the end of the first third of embryonic development, new capillaries, including those which arise later in the embryo and in the adult, are derived from existing vessels. It is this angiogenesis or neovascularization phenomenon to which the invention is applied.
The complete process of angiogenesis is not entirely understood, but it is known to involve the endothelial cells of the capillaries in the following ways:
1) The attachment between the endothelial cells and the surrounding extracellular matrix is altered, presumably mediated by proteases and glycosidases which may be produced at enhanced levels during this process;
2) There is a "chemotactic" process of migration of the endothelial cells toward the tissue to be vascularized; and
3) There is a "mitogenesis" process--i.e., proliferation of the endothelial cells to provide additional cells for the new vessels.
Each of these activities involved in angiogenesis: proteolysis, chemotaxis, and mitogenesis, can be measured independently in in vitro endothelial cell cultures. In addition, the overall angiogenic stimulation of a test substance can be measured in model systems such as the chick chorioallantoic system (which measures angiogenic activity in an embryonic system), and in the rabbit corneal pocket assay and in hamster cheek pouch assay (which measure angiogenic activity in more mature systems).
A number of factors are known to stimulate angiogenesis. Many of these are peptide factors, and the most notable among these are the fibroblast growth factors, both acidic and basic which can be isolated from a variety of tissues including brain, pituitary, and cartilage. A discussion of various peptide factors involved in angiogenesis, including acidic and basic FGF is found in, for example, PCT application WO87/01728 published Mar. 26, 1987 and incorporated herein by reference. Both isolated and recombinant forms of many of these proteins are now available.
Other factors which are known to show angiogenic-stimulating activity, but which are not proteins, include prostaglandins E1 and E2 (Ben-Ezra, D., et al., Am J Opthamol (1978) 86:445-461) fragments of hyaluronic acid (West, D. C., et al., Science (1985) 228:1324-1326) and nicctinamide (Kull, F. C., et al., Science (1987) 236:843-845).
A series of papers published by groups from Boston University and Angio Medical Corporation led by N. Catsimpoolas, describe the isolation of a lipid angiogenic factor from omentum. A preliminary communication appeared in JAMA (1984) 252:2034-2036. There are also a number of patent publications from this group, including PCT applications WO87/03804, WO87/03811 and WO87/03812, all published Jul. 2, 1987; WO87/03486, published Jun. 18, 1987; WO87/06136, published Oct. 22, 1987; and U.S. Pat. No. 4,699,788, issued Oct. 13, 1987. These applications describe and claim use of compositions of omental lipids or fractions for skin care and cosmetics or for angiogenesis and epithelialization.
In addition, conditioned media from 3T3 adipocytes, but not from undifferentiated 3T3 cells, has been shown to stimulate angiogenesis related processes in endothelial cells (Castellot, J. J., Jr., et al., Proc Natl Acad Sci USA (1980) 77:6007-6001). This paper described mitogenic activity for endothelial cells in vitro exhibited by the 3T3 adipocyte culture medium which was insensitive to proteasee, not inactivated by heat, and dialyzable.
Further work by this same group, which includes the inventors herein, showed that these conditioned media (but not the medium conditioned by preadipocytes) could stimulate angiogenesis in the chicken chorioallantoic membrane (CAM) model, and that this stimulation was potentiated by heparin. (Castellot, J. J., Jr., et al., Proc Natl Acad Sci USA (1982) 79:5597-5601). The conditioned medium was also shown to effect chemotaxis and motility in the Boyden chamber assay described by Zigmond, S., et al., J Exp Med (1973) 137:387-410; Postlethwaite, A. E., et al., J Exp Med (1976) 144:1188-1203; and by Grotendorst, G. R., et al., Proc Natl Acad Sci USA (1981) 78:369-372, all incorporated herein by reference.
Further characterization of the heparin potentiation of the adipocyte-stimulated angiogenesis was reported by the same group (Castellot, J. J., Jr., et al. (1986) 127:323-329. This work showed that both anticoagulant and nonanticoagulant heparin were capable of potentiation of the activity of the 3T3 adipocyte-conditioned media in the CAM assay.
When the three aspects of angiogenesis separable in vitro were tested, it was found that heparin potentiated the effect of the conditioned media on protease activity (measured as pasminogen activator activity) and motility but did not affect the mitogenic activity of the medium. The effect of heparin on the protease activity depended on the nature of the protease-heparin stimulated tissue plasminogen activator activity, but inhibited urokinase activity.
The present invention provides purified and synthetic factors capable of angiogenesis which are suitable for pharmaceutical formulation and administration. These factors are members of a group of compounds which are low molecular weight monoglycerides.